6022911NCGCRXFP808201322qHTS Assay for Agonists of the Relaxin Receptor RXFP1: Cell ImpedanceThe relaxin hormone is involved in the variety of biological functions in normal tissues and diseases. The role of relaxin is well-established in female reproduction and parturition, mammary gland and endometrial development, maintenance of myometrial quiescence during pregnancy. Relaxin signaling through its G protein-coupled receptor (GPCR) RXFP1 results in ECM remodeling through regulation of collagen deposition, cell invasiveness, proliferation and overall tissue homeostasis. Significantly, the therapeutic effects of relaxin in the treatment of renal, cardiac, skin, lung fibrosis, inflammation, and wound healing in animal models are well-established. Recombinant human relaxin (rhRlx) is currently being tested in clinical trials as a protective agent in congestive heart failure, in treating severe preeclampsia, and as an anti-fibrotic agent in systemic sclerosis.Upon relaxin binding RXFP1 activation leads to the activation of adenylate cyclase (AC) via Gs. cAMP will activate PKA, which phosphorylates many signaling proteins. Thus, detection of cAMP increase is an easy and reliable indication of relaxin receptor activation. To screen for agonists of the relaxin receptor, a HEK293T cell line stably transfected with RXFP1 was used. RXFP1 activation was assayed by changes in cAMP levels as detected with a time-resolved fluorescence energy transfer (TR-FRET) cAMP detection kit.After the primary screen and rounds of SAR, a probe and analog were identified. It was previously shown that relaxin increases cell impedance in RXFP1 transfected cells. A cell line stably transfected with RXFP1 receptor HEK293-RXFP1 was used for cell impedance assay to confirm relaxin-like properties of the compounds. NIH Chemical Genomics Center [NCGC]NIH Molecular Libraries Probe Production Centers Network [MLPCN]MLPCN Grant: R03 MH085705Assay Submitter (PI): Alexander AgoulnikTo equilibrate the plates, 100 microL of test media (DMEM, 1% FBS, 1x Pen/Strep) was added to each well of E Plate 16 (Roche Diagnostics, Indianapolis, IN) and the plate was incubated at room temperature for 30 minutes at which point baseline impedance was measures. Then 20,000 HEK293-RXFP1 cell or HEK293 cells (parental control cell line) were added per well in a volume of 100 microL test media and allowed to sediment at room temperature for 30 minutes. The plate was placed into xCELLigence RTCA DP Instrument in the CO2 incubator overnight to allow the cells to attach. Relaxin (10 ng/mL), vehicle, or compounds 500 nM were added to the wells and the cellular impedance was measured every 10-30 seconds for 1 hour.Compounds are considered "active" if an increase of cell impedance is observed; compounds are considered "inactive" if no increase in cell impedance is observed.A PubChem Activity score is assigned based on an increase of activity. Relaxin is assigned a score of 100 and inactive compounds are assigned a score of 0.2703Summary AID59350RXFP1 relaxin/insulin-like family peptide receptor 1 [ Homo sapiens ] 11 min500 nM115210 min500 nM115320 min500 nM115430 min500 nM115540 min500 nM1156Compound QC42541relaxin receptor 1 isoform 1 [Homo sapiens]859866011Homo sapienshumantaxon96060MH0857052994454971210014.276315789233.46052632375.10526316493.47368421598.881578956QC'd by "NCGC"2012351342250881267119.18421053241.71052632356.02631579462.552631585676QC'd by "NCGC"201235134225113124017.592105263212.42105263326.75434.15789474539.486842116QC'd by "NCGC"201235134225124123110.0142857142-0.771428571312.88571429424.14285714531.228571436QC'd by "NCGC"201235134225125124612.84285714326.985714286322.5438.85714286546.671428576QC'd by "NCGC"201235